Depression and addiction affect millions of individuals each year exerting untold costs on society. The dopamine transporter (DAT), a membrane protein that clears dopamine (DA) released into the extracellular space is a target of clinically used antidepressants and several drugs of abuse. ?-opioid receptors (KOR) are enriched in brain circuits that subserve mood and motivation and regulate the basal activity of DA neurons located therein. Upregulation of KOR systems has been implicated in the pathogenesis of depression and the mood dysregulation that characterizes withdrawal from cocaine and other drugs of abuse. Despite on-going efforts to develop orally effective KOR ligands for the treatment of depression and addiction, the downstream effectors upon which these agents act to affect behavior and DA transmission are unknown. The dysphoric and aversive effects of KOR agonists have been attributed to decreased DA release in the ventral striatum (VST). Importantly, however, KOR in VST are apposed to the dopamine transporter. Our studies show that KOR activation increases DAT activity through ERK1/2 dependent phosphorylation of threonine (Thr)53 of DAT and selective inhibition of ERK1/2 in the VST attenuates the aversive effects of KOR agonists. These findings identify a novel mechanism by which KOR ligands regulate presynaptic DA transmission and suggest that transporter dysregulation may be one mechanism underlying KOR-mediated alterations in mood and affect. The studies in this proposal will test the hypotheses that: KOR activation modulates DA dynamics and VST-dependent behaviors via ERK1/2-dependent phosphorylation and regulation of DAT. Specific Aim 1 will identify the cellular mechanisms of KOR-linked DAT modulation by determining whether KOR-mediated changes in DAT function and expression in the VST are associated with Thr53 phosphorylation and whether manipulations that prevent KOR-agonist evoked ERK1/2 activation and DAT phosphorylation in the VST attenuate KOR-mediated changes in DAT function. Specific Aim 2 will establish the relevance of this mechanism to the regulation of presynaptic DA transmission by determining whether manipulations that prevent KOR-agonist evoked ERK1/2 activation and DAT phosphorylation alter basal DA dynamics. Specific Aim 3 will determine the physiological relevance of KOR-ERK1/2 linked DAT modulation to the behavioral effects of KOR agonists and antagonists by assessing whether prevention of KOR-ERK1/2 linked DAT modulation in the VST attenuates the aversive and pro-depressive like effects of KOR agonists as well as the antidepressant-like effects of KOR antagonists. The role of KOR-ERK1/2 linked DAT modulation in mediating the efficacy of KOR agonists in preventing the locomotor stimulant effects of cocaine will also be assessed. Findings from these studies will enhance our understanding of the neural substrates upon which KOR ligands act to regulate mood and DA transmission. Furthermore, they will provide new insights as to the role of DAT phosphorylation in regulating synaptic DA clearance and behavior. PUBLIC HEALTH RELEVANCE: Kappa-opioid receptors are enriched in brain circuits that subserve mood and motivation and regulate the basal activity of both dopaminergic and serotoninergic neurons located therein. The proposed studies will test the hypothesis that kappa-opioid receptors affect monoamine transmission by activating kinase cascades that regulate the function of dopamine transporters. Outcomes from the proposal will enable identification of the molecular targets by which ?-opioid receptors regulate dopamine transmission and aid in the development of effective pharmacological agents for the treatment of addiction and other disease states resulting from aberrant monoamine transmission.